The present invention relates to a method for performing cycled primer extension on a DNA template, and more particularly to methods including a primer extension step such as polymerase chain reaction (PCR) amplification and nucleotide sequencing comprising performing said PCR and sequencing reactions in the presence of an osmoprotectant selected from proline 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine (hereinafter xe2x80x9cTHP(B)xe2x80x9d), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine (hereinafter xe2x80x9cTHP(A)xe2x80x9d) and mixtures thereof, to improve yield and specificity of said reactions. The invention further relates to kits comprising proline, THP(B), THP(A), or mixtures thereof for use in PCR amplification and in cycle nucleotide sequencing.
Primer extension on a DNA template is a step common to some of the most useful and powerful techniques in molecular biology. Polymerase chain reaction (PCR), one of these techniques, is a rapid, inexpensive and simple means of producing microgram amounts of DNA from minute quantities of source materials. Many variations on the basic procedure have now been described and applied to a range of disciplines.
In medicine, PCR""s major impact is on the diagnosis and screening of genetic diseases and cancer, the rapid detection of mycobacteria and HIV, the detection of minimal residual disease in leukemia, and HLA typing. The PCR technique is also useful in forensic pathology and evolutionary biology, plays a central role in the human genome project and is routinely used in molecular biology processes (McPherson et al., 1992).
However, the practical use of PCR technology frequently faces difficulties and limitations. The necessity to convert originally duplex source DNA and then double-stranded DNA products into single stranded templates in every cycle of amplification is normally accomplished by thermal denaturation of DNA at 93-95xc2x0 C. The DNA denaturation greatly depends on its nucleic base composition. A high GC content renders DNA amplification and sequencing very difficult, due to increased melting temperature and the stable secondary structure of the expanded motif. A common result of amplifying a region containing a repeat motif with a high GC content is the presence of additional amplification products, which do not correspond to the desired product (Varadaraj and Skinner, 1994). In addition, incomplete denaturation allows DNA strands to xe2x80x9csnap backxe2x80x9d, leading to a decrease in product yield. Denaturation steps that are conducted for long periods of time and/or at a high temperature lead to unnecessary loss of enzyme activity and dNTP decomposition.
Taq DNA polymerase, ordinarily used in PCR protocols, can withstand repeated exposure to the high temperature (94-95xc2x0 C.) required for typical DNA strand separation, and thus simplifies the PCR procedure by eliminating the need to add an enzyme in each cycle. However, Taq polymerase appears to extend a mismatched primer/template in comparison to other polymerases with proofreading exonuclease activities, e.g. Klenow and T7 DNA polymerases, which are non-thermostable.
Another very effective technique employing primer extension is the cycle sequencing technique used for determining the order of nucleic acids in a target nucleotide sequence. This procedure involves repeated cycles of primer extension while the target nucleotide sequence is sequenced.
Similar considerations, as mentioned above for the PCR method, apply for the cycle sequencing procedure. In sequencing reactions as well, the complete denaturation of the template DNA is of crucial importance for a successful reaction. Thus, regions of DNA with repeat motifs, high GC content and rigid secondary structures are difficult to sequence. In addition, sequencing of a very long stretch of nucleotides, or of a target nucleotide sequence present in a minute amount is problematic. The ability to accomplish a complete denaturation of double stranded DNA and to perform sequencing reactions at reduced temperatures, either with Taq polymerase or with non-thermostable polymerase, is advantageous in terms of both yield and accuracy.
In an attempt to improve the yield and specificity of PCR and sequencing reactions, a number of buffer additives were employed. It was shown that certain cosolvents, such as DMSO (Pomp and Medrano, 1991; Filichkin and Gelvin, 1992), glycerol (Cheng et al., 1994; U.S. Pat. Nos. 5,432,065 and 5,545,539), formamide (Comey et al., 1991) and betaine (German Patents DE 4411594 C1 and DE 4411588 C1; Mytelka et al., 1996), facilitate standard PCR and/or cycle sequencing. It has been suggested that DMSO may affect the melting temperatures (Tm) of the template DNA and of the oligonucleotide primers and/or the degree of product strand separation at a particular xe2x80x9cdenaturationxe2x80x9d as well as improving the thermal activity of Taq DNA polymerase (Gelfand and White, 1989). Glycerol may influence long amplifications by (i) doubling the thermal stability of Taq polymerase at 95-97xc2x0 C., and (ii) effectively lowering DNA melting temperatures (by 2.5-3xc2x0 C. for each 10% increase in glycerol concentration) (Cheng et al., 1994). Yet, the use of these buffer additives is limited, e.g. solutions containing glycerol in effective concentrations of 20-40% are viscous and difficult to handle (U.S. Pat. No. 5,432,065), DMSO in 10% concentration inhibits Taq DNA polymerase activity by 53% and T7 DNA polymerase is completely inactive in 40% formamide.
The compounds 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)], also known as ectoine, and its hydroxy derivative, 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] were previously identified in the laboratory of the inventors of the present invention as metabolites in several Streptomyces microorganisms (Inbar and Lapidot, 1988a; 1988b and 1991; Malin and Lapidot, 1996). Ectoine was also found in a variety of halophilic and halotolerant bacteria (Galinski et al., 1985). THP(B) and THP(A) are zwitterionic compounds (Inbar et al. 1993; FIG. 1) with many useful properties such as osmoprotection and thermoprotection of several organisms of the Streptomyces species and E. coli cells (Malin and Lapidot, 1996). THP(B) and THP(A) are not toxic neither to mammalian cells nor to animals (Lapidot et al., 1995). Israel patent No. 100810 and corresponding U.S. Pat. No. 5,789,414 and European Patent No. EP 0553884 of the present applicants disclose that THP(A) and THP(B) interact with and protect DNA in non-tumor tissues from damage by DNA-binding drugs and thus can be used for decreasing the toxic effects of DNA-binding drugs such as adriamycin and actinomycin D.
Proline is another osmoprotectant that accumulates in plants, bacteria, algae and marine invertebrates as a response to salinity stress. Proline was shown to destabilize DNA and to partially counteract the effect of sodium chloride and spermidine on the stability of the double helix, and to lower the melting temperature of DNA in a concentration-dependent manner (Rajendrakumar et al., 1997).
None of the above references describes or suggests the use of proline, THP(A) or THP(B) or mixtures thereof as additives to PCR reaction mixtures and in reactions for nucleotide sequencing.
It has now been found, according to the present invention, that THP(B) is effective in lowering the melting temperature of double-stranded DNA, and that proline, THP(B) and THP(A) are capable of increasing the thermal stability of DNA polymerases at elevated temperatures, indicating that they can be useful in procedures involving melting of double-stranded DNA and/or polymerase-mediated DNA synthesis, such as in primer extension, in PCR (polymerase chain reaction) amplification and in DNA sequencing.
Thus, in one aspect, the present invention provides a method for performing a cycled primer extension reaction comprising the steps of:
(i) contacting a template DNA comprising a target sequence of nucleotides with at least one primer oligonucleotide complementary to a nucleotide sequence at the 3xe2x80x2-end of said target sequence, under conditions allowing annealing of said primer to its complementary nucleotide sequence on said target sequence, in the presence of an osmoprotectant selected from proline, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)] and mixtures thereof, to lower the melting temperature of said template DNA and/or of said primer; and
(ii) carrying out a polymerase-mediated extension of said primer on said target sequence of nucleotides in the presence of an osmoprotectant selected from proline, THP(B), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] and mixtures thereof, to stabilize said polymerase,
thus obtaining a high yield specific extension of the primer on said target sequence of nucleotides of the template DNA.
Steps (i) and (ii) may be repeated a plurality of times, for example 10-90 times, preferably 15-35 times, and each step (i) is preceded by DNA thermal denaturation at a temperature suitable for separating both said template DNA into its strands and the polymerase-extended primer of step (ii) from its complementary target sequence of nucleotides, said temperature being a temperature in which the polymerase used in step (ii) is stable.
In one embodiment, the invention relates to a method for determining a nucleotide sequence of a target DNA, wherein in step (i) the target sequence of the template DNA is a sequence of nucleotides to be sequenced, and the polymerase-mediated extension of the primer in step (ii) is carried out in the presence of all four dNTPs: dATP, dCTP, dGTP and dTTP, and in the presence of a minute amount of either ddATP, ddCTP, ddGTP or ddTTP, prior to the determination of the nucleotide sequence of the target DNA. The dGTP can be substituted by 7-deaza-dGTP described in EP 0212536.
According to this embodiment, the method for determining a nucleotide sequence of a target DNA comprises the steps of:
(i) heating a template DNA comprising a target sequence of nucleotides to be sequenced at a temperature suitable for separating said template DNA into its strands in the presence of an osmoprotectant selected from proline, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)] and mixtures thereof;
(ii) contacting said denatured template DNA of step (i) with a primer oligonucleotide complementary to a nucleotide sequence at the 3xe2x80x2-end of said target sequence of nucleotides under conditions allowing annealing of said primer to its complementary nucleotide sequence on the target sequence, in the presence of an osmoprotectant selected from proline, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)] and mixtures thereof;
(iii) carrying out a polymerase-mediated extension of said primer of step (ii) in the presence of all four natural dNTPs: dATP, dCTP, dGTP (or 7-deaza-dGTP) and dTTP, of a minute amount of either ddATP, ddCTP, ddGTP or ddTTP and of an osmoprotectant selected from proline, THP(B), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] and mixtures thereof;
(iv) repeating steps (i)-(iii) a plurality of times; and
(v) determining the nucleotide sequence of the target DNA.
In another embodiment, the invention provides a method for amplifying a target sequence of nucleotides by polymerase chain reaction (PCR), wherein in step (i) the target sequence of the template DNA is a sequence of nucleotides to be amplified and the template DNA is contacted with two oligonucleotide primers complementary to the nucleotide sequences at the 3xe2x80x2-ends of said target sequence of nucleotides and its opposite strand; in step (ii) a polymerase-mediated extension of the annealed primers of step (i) is carried out; steps (i)-(ii) are repeated a plurality of times, the last step being step (ii), thus generating multiple copies of the target sequence of nucleotides.
According to this embodiment, the invention relates to a method for amplifying a target sequence of nucleotides by polymerase chain reaction (PCR) comprising the steps of:
(i) heating a template DNA comprising a target sequence of nucleotides to be amplified at a temperature suitable for separating said template DNA into its strands in the presence of an osmoprotectant selected from proline, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)] and mixtures thereof;
(ii) contacting the template DNA of step (i) with two oligonucleotide primers complementary to nucleotide sequences at the 3xe2x80x2-ends of said target sequence of nucleotides and its opposite strand; in the presence of an osmoprotectant selected from proline, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)] and mixtures thereof, under conditions allowing annealing of said oligonucleotide primers to their complementary sequences on said target sequence of nucleotides and its opposite strand;
(iii) carrying out a polymerase-mediated extension of the annealed primers of step (ii) in the presence of an osmoprotectant selected from proline, THP(B), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] and mixtures thereof; and
(iv) repeating steps (i)-(iii) a plurality of times, the last step being step (iii), thus generating multiple copies of the target sequence of nucleotides.
The methods of the invention are particularly useful for reactions involving GC-rich DNAs, thus diminishing or eliminating the difficulties found in amplification and sequencing of GC-rich DNA molecules. The methods are further useful for reactions involving in step (ii) or (iii) a thermostable DNA polymerase, such as Taq polymerase Klentaql polymerase and Pfu polymerase, or a non-thermostable DNA polymerase such as T7 DNA polymerase, T4 DNA polymerase, Klenow fragment of DNA polymerase I, reverse transcriptases, Bca polymerase, Bst polymerase and mutants of these polymerases.
In another aspect, the invention relates to the use of an osmoprotectant selected from proline, THP(B), THP(A) and mixture thereof as an additive in a reaction for determining a nucleotide sequence or as an additive to a PCR reaction mixture, and to kits comprising in separate containers: (a) the reagents necessary for DNA sequencing or the reagents necessary for a polymerase chain reaction, and (b) proline, THP(A) or THP(B).
In a further aspect, the invention relates to a method for lowering the melting temperature of double-stranded DNA (dsDNA) comprising adding to the incubation mixture of said dsDNA an effective amount of THP(B).
In a further aspect, the invention relates to a method for increasing stability of a DNA polymerase at elevated temperatures comprising adding to the incubation mixture of said polymerase an effective amount of an osmoprotectant selected from proline, THP(B), THP(A) and mixtures thereof.